Photonic sensors, xerogel-based sensors and nanosensors

ABSTRACT

A photonic sensor system is provided. The system generally includes a beta emission source, optionally, a scintillation layer, and a luminophore-containing sensory layer. The system can be embodied in a particle. Also provided are photonic sensor strategies which are highly accurate and photonic sensors which are highly stable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/076,729, filed Mar. 10, 2005, which claims priority to U.S.Provisional Application Ser. No. 60/551,818, filed on Mar. 10, 2004, thedisclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant numbersCHE-0315128 from the National Science Foundation (NSF) andN00014-02-1-0836 from the Office of Naval Research (ONR). The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to area of detection ofanalytes.

BACKGROUND OF THE INVENTION

The desire to simultaneously measure “everything” in any sample hasdriven the development of artificial “noses” and “tongues”. Thesedevices have been based on a wide variety of sensor array strategies.Examples of sensor arrays include those based on cantilevers, conductingpolymers, electrochemistry, photonics, the piezoelectric effect, orsurface acoustic waves. In a photon-based sensor, one requires a photonsource, appropriate sensing chemistry (i.e., a recognition element), ameans to immobilize the desired sensing chemistry so it may be reused,and a photon detector. All reported photon-based sensor arrays depend onat least one photon source (e.g., lamps, lasers, light emitting diodes)and all currently used photon sources in photon-based sensor arraysrequire electrical power. This need for electrical power limits anyphotonically-based sensor device.

Although a number of strategies exist for developing photonically-basedsensor arrays (vide supra), there is clearly a need for new strategiesthat use little or no electrical power. Specifically, there is a needfor devices wherein the photon/light source operates without anyexternal electrical power. Hieftje and coworkers (Rev. Sci. Instrum.1999, 70:50-57) introduced the idea of using a beta emitter, ⁹⁰Sr, and aliquid or plastic scintillator in concert with time-correlated singlephoton counting as a pulsed photon source for the determination ofexcited-state fluorescence lifetimes.

A system has been developed to address the limitations of existingsensor array photon sources. Our source system can potentially work withany photonically-based chemical sensor modality. It consists of a sealed⁹⁰Sr beta emitter (with or without a scintillator), a chemicalresponsive sensor array, and an array-based photodetector. It can serveas part of a complete photonically-based sensor array system that hastwo major features. First, the photon source does not require anyelectrical power and the source can be operated continuously, in anunattended manner, for several years. Thus, electrical power is onlyneeded to power the photodetector in this system. Second, the so formedphotonic sensor array is capable of the detection and/or quantificationof more than one analyte in a sample at the same time.

SUMMARY OF THE INVENTION

The present invention provides a photonic sensor system for thedetection of, and in one embodiment, the two dimensional resolution of,a chemical analyte. It functions by measuring the radiation from sensorswhich are in contact with a sample. The sensors are stimulated toradiate either by a beta emission source or a by radiation from ascintillation layer which is interposed between a beta emission sourceand the sensor. During exposure of the sensors to the desired analyte,the radiation from the sensor changes in intensity, wavelength or both.By comparing the radiation during analyte exposure to the radiation fromthe sensor unexposed to the analyte, the concentration of analyte in asample can be determined. If a sensory array is used, two dimensionalresolution of concentration can be determined. Distributions of sensorscan be used to give an “image ” of analyte in a sample. If a sensoryarray with sensors which are sensitive to multiple different analytesare used, multiple analytes can be simultaneously detected in a sample.Also provided are sensory arrays which have increased reliability andlower error than existing sensors due to the use of an array withdiversified sensors for a single analyte.

In general, unlike existing methods, the source of photons which impingeon the sensory array (i.e., the beta emission source, either with orwith out a scintillation layer) does not require an external powersource, and can remain stable over long periods of time withoutattention.

The present invention further provides a method for preparing sensoryarrays which allow greater spatial resolution than previouslyattainable. The method comprises the creation of pin printing arrays inwhich the pins have been created from specific materials by pulling,cutting or etching, optionally followed by the treatment of the pin tipswith materials which allow the easy dispensing of ultra-small arraysensors.

As an embodiment of the sensor systems described above, also providedare nanosensors which have integrated beta emission sources and asensory layer analogous to a sensor. These nanosensors are introduceddirectly into the sample, and the radiation from the sensory layers ofthe particles is collected and analyzed to determine an analyte profile.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified schematic of the beta emitter emitter-based senorarray system.

FIG. 2 shows examples of possible beta emitter-scintillator-sensorelement geometries.

FIG. 3 is a false color image of a [Ru(dpp)₃]²⁺-doped xerogel-basedsensor array when excited by the combination of a 2 mCi⁹⁰Sr beta emitterand an EJ-200 scintillator. The diameter of a typical sensor element is˜200 μm.

FIG. 4 is a typical calibration curve for an O₂ responsive sensorelement excited by the combination of a 2 mCi⁹⁰Sr beta emitter and anEJ-200 scintillator.

FIG. 5 represents imaging strategy which uses with intrinsic reportersor extrinsic sensors.

FIG. 6 shows an illustration of a one-photon excitation strategy.

FIG. 7 shows an illustration of a multi-photon excitation strategy.

FIG. 8 shows an illustration of nanosensor particle structure (top left)and the sequestering, in specific sample domains, of particles whichcomprise a directing layer.

FIG. 9 is a depiction of exemplary beta emitter-sensing layerconfigurations.

FIG. 10 is a depiction of exemplary changes in sensor response uponexposure of the sensor to an analyte. A) shift in emission; b) decreasein intensity of emission; c) change in emission lifetime.

FIG. 11 is a diversification of sensors. A) examples of differentsensing chemistries/sensing layer formulations for the same targetanalyte; 11b) examples of calibration curves 1-3 representing differentsensing chemistries/sensing layer formulations for the same targetanalyte. Potential calibration curves are presented in FIG. 11 c.

FIG. 12 is a schematic of the pin system along with eight possible pintypes/geometries.

FIG. 13 represents potential geometries at the distal end of achannel-based pin.

FIG. 14 represents examples of pin printed microarrays formed with thenew pins compared to quill, and 200, 400 and 600 μm solid pins. Featuresizes are shown.

FIG. 15 shows a portion of a diversified, xerogel-based sensor library.The xerogel composites are identical. Sensors a-e contain differentmolar ratios of the dpp and bpy-based luminophores. This is an exampleof τ₀-based diversification. The sensor element feature size is ˜100 μmin diameter.

FIG. 16 shows calibration curves for the sensor elements shown inFIG. 1. (panel A) gaseous O₂. (panel B) dissolved O₂.

FIG. 17 shows calibration curves for another type of diversified,xerogel-based sensor library. The luminophore is identical in thesesensor elements. The individual sensors are derived from equal molarmixtures of c_(n)-TMOS and TMOS or c_(n)-TEOS and TEOS. This is anexample of kq-based diversification.

FIG. 18 shows low-resolution SEM images of xerogel films. The TEOS filmis 2 months old; all other films are 3 months old. Film age does notaffect the SEM images.

FIG. 19 shows high-resolution SEM images of various regions within atypical 60 mol % octyl-triEOS/40 mol % TEOS composite xerogel filmshowing domain separation and heterogeneity. These films are 3 monthsold. Film age does not affect the SEM images.

FIG. 20 shows effects of storage/aging time and xerogel composition onthe average o₂ sensitivity.

FIG. 21 shows typical intensity-based Stern-Volmer plots for 3-month old[Ru(dpp)₃]²⁺-doped octyl-triEOS/TEOS composite xerogels. The solid linesrepresent the best fit to a Dernas (TEOS) or Stern-Volmer model (allothers).

FIG. 22 shows effects of xerogel composition on the average Stern-Volmerquenching constant. The films are 3 months old.

FIG. 23 shows typical time-resolved intensity decay traces and fits(lines) for 3-month [Ru(dpp)₃]²⁺-doped octyl-triEOS/TEOS compositexerogels in a pure N₂ environment. The [Ru(dpp)₃]²⁺-doped intensitydecay is clearly biexponential for the pure TEOS and 20 mol %octyl-triEOS composite xerogels. The [Ru(dpp)₃]²⁺ intensity decay issingle exponential in those xerogels that contain >20 mol %octyl-triEOS.

FIG. 24 shows effects of xerogel composition on the average [Ru(dpp)₃]²⁺excited-state fluorescence lifetime <<τ>=Σ(Fτ) and the bimolecularquenching constant. The films are 3 months old.

FIG. 25 is a simulated i₀/i vs. [q] plot for a sensor element thatcontains two luminophores with excited-state lifetimes of 5000 ns and500 ns.

FIGS. 26-31 show results from a series of simulations based on the samesensor element.

FIGS. 32-34 show three-dimensional simulations for the sensor element ofFIG. 25.

FIG. 35 shows use of a sensor element containing a xerogel doped with[Ru(dpp)₃]²⁺ and [Ru(bpy)₃]²⁺ and excited with 450 nm light that ismodulated at 20 kHz.

DETAILED DESCRIPTION OF THE INVENTION

Provided by the present invention are photonic sensor arrays based on anon-electrical light source. A view of the invention is given in FIG. 1.The source system consists of a sealed ⁹⁰Sr beta emitter housed within aceramic disc which is in turn incorporated within a stainless steel bodyalong with an optional scintillator and an array of photonically activesensor elements. The sequestration of the ⁹⁰Sr within the ceramic matrixin conjunction with the stainless steel capsule precludes anyradioactive material leaching. Other radioactive sources could be used.Such a source is commercially available from AEA Technology, QSAIncorporated (Burington, Mass.). The beta radiation from the sealed ⁹⁰Sremitter is directed toward the chemical sensor array. Otherbeta-emitters can also be used in the photonic sensor system of thepresent invention, such as ³H, ¹⁴C, ²²Na, ³²Si, ³²P, ³⁵S, ³⁶Cl, ⁴¹Ca,⁵⁷Co, ⁶⁰Co, ⁶³Ni, ⁸⁹Sr, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Tc, ¹²⁹I, ¹³⁷Cs, ¹⁴⁷Pm, ¹⁵¹Sm,²⁰⁴Tl, ²¹⁰Pb, ²³⁷U, ²³⁸Np, or ²⁴¹Pu.

While not required, in some embodiments, between the source and thesensor array is a scintillation medium, such as, for example, a liquidscintillator solution or a plastic scintillator material. A wide varietyof scintillation materials can be used, such as, for example, EJ-200,EJ-204, EJ-208, EJ-212, EJ-232, EJ-301, EJ-321, EJ-331, and EJ335 fromEljen or similar products from sources like Zinsser Analytic orAmcrys-H. In general, the choice of scintillation material between thebeta emitter and the array of sensor elements depends on the chemicaland photonic nature of the sensing chemistry and the associatedluminophores in that radiation emitted by the scintillation materialshould be comprised of a wavelength that the luminophores present in thesensor can absorb.

In one embodiment, the beta particles are emitted from the sealed sourceand they impinge upon scintillator molecules which produce photons,these photons impinge upon the sensors, exciting luminophores containedwithin the sensors. In another embodiment, the beta particles impingedirectly upon the luminophores sequestered within the sensor elements(FIG. 1). The presence of a scintillating layer gives increasedflexibility to the system because it is generally possible to choose ascintillator which produces a wavelength of light which corresponds toan absorption wavelength of the chosen luminophore or chromophore.Regardless of which embodiment is used, the resulting emission (orabsorbance) from the sensor elements is detected by one of many possiblearray-based photon detectors (e.g, charge transfer device, complementarymetal oxide semi conductor) known to those familiar with the disciplineof photon detection.

Luminophores or chromophores which can be used in the process photonicsensor system of the present invention absorb or emit in theultraviolet, visible or infrared. Non-limiting examples of reporterswhich can be used include luminescent organic or inorganic species likefluorescein, BODIPY, rhodamine; organometallic complexes liketris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺; andluminescent nanoparticles (i.e., quantum dots). Non-luminescent dyemolecules that are responsive to their physicochemical environments canalso be used (e.g., 4-nitroaniline, and2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt's dye30), 2,6-dichloro-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt'sdye 33), and N,N-diethyl-4-nitroaniline).

In general, the sensors in the present invention can be of any typewhich comprise luminophores or chromophores which give changes in theirabsorbance and/or emission spectra, polarization, and/or excited-statelifetime upon exposure to the analyte for which a sensor is desired.Examples of types of sensors which can be used in the present inventionare molecularly imprinted polymers, gels and xerogels; monolayer-basedsensor elements, protein-based biosensors and others known to thoseexpert in the field of chemical sensing. Analytes which can be detectedare diverse, including molecules as small as oxygen to molecules aslarge as proteins and peptides.

Solid scintillation materials have the advantage that a sensor or sensorarray can be formed on the face of the scintillator, or within thescintillator as shown in FIG. 2.

The absorbance from the sensor elements within the array are passedthrough collection optics, an optical filter, and detected by a suitablearray detector (e.g., a charge transfer device or a complementary metaloxide semiconductor). The signals from the photodetector can also beprocessed by a variety of methods, including, for example, on-chipelectronics or within a computer.

The present invention is not limited in scope to the measurement ofsensor emission. It should be noted that other sensor properties, suchas changes in absorption of radiation (instead of changes in emission)due to the exposure of the sensor to an analyte can be measured andprocessed to give the benefits of the invention described herein.

The use of an array can also give a spatially resolved image of theconcentration of a particular analyte. FIG. 3 presents a false colorimage from a portion of an O₂ responsive sensor array that was preparedfrom a binary nanoporous xerogel glass composed of 1:1 mole ratio oftetramethyorthosilane (TMOS) and octyl-trimethoxysilane (C8-TrMOS) dopedwith an O₂ responsive luminophore(tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dication,[Ru(dpp)₃]²⁺). In the presence of O₂ the [Ru(dpp)₃]²⁺ emission isquenched in step with the O₂ concentration. (A calibration curve(Stern-Volmer plot) for these sensor elements when they are excited bythe combination of a single 2 mCi ⁹⁰Sr beta emitter and an EJ-200scintillator is presented in FIG. 4.)

Also provided by the present invention is method of imaging whichdiffers from current methods.

There are numerous problems that require one to identify and determinethe concentration of a particular molecule (i.e., the target analyte)within a sample/specimen in a spatially defined manner. Examples includethe detection of a specific target analyte within polymer-basedcomposites, living cells, or tissues. One common strategy (shown in FIG.5) is to use luminescence in concert with intrinsic reporters orextrinsic sensors. Examples of intrinsic reporters include luminescentspecies like the aromatic residues attached to certainoligomers/polymers, proteins (aromatic amino acids or other chromophorestherein), reduced nicotine adenine dinucleotide (NADH), flavins, andbilirubin. These species are intrinsic to a particular system/specimen,they are luminescent, and their emission can be used to map theirlocation within the system/specimen. Autoemission—can also haveintrinsic diagnostic value. For example, sample/specimenautofluorescence spectra have been used to screen for bladder,colorectal, lung and skin cancers, to assess dental hard tissue, andscreen foods. An extrinsic sensor is added to the system by one of manystrategies (e.g., physical mixing, gene gun injection into a cell) andthe tiny sensors distribute themselves within the sample/specimen andthey report on their target analyte. [Note: This distribution processcan be made pseudo selective by using selective biomolecules (e.g.,antibodies) to deliver the sensors to a specific site within thesample/specimen (e.g., interface, mitochondria).]

If the sensor shown in Domain 1 of FIG. 5 were designed to detect targetanalyte A, the concentration of A at that specific site on the edge ofDomain 1 could be determined. Similarly, the sensor shown in Domain 3 ofFIG. 5 could report on analyte B within Domain 3. The idea of“injecting” sensors into a sample/specimen is at the heart of the socalled nanopebble-based photonic sensors developed by the Kopelman group[cf., Park, E. J. et al. Anal. Chem. 2003, 75, 3784-3791 and referencescited therein]. A related example involves the use of selective orsemi-selective synthetic chromophores/luminophores that respond to aspecific non-fluorescent target analyte (e.g., Calcium Green for Ca²⁺,fluorescein for pH, IndoZn for Zn²⁺).

Typical optical configurations for exciting the luminescent reportersand/or sensors are shown in FIGS. 6 and 7. FIG. 6 represents aone-photon excitation situation and FIG. 7 means to illustrate amulti-photon excitation strategy. Regardless of the case, the currentstate-of-affairs requires that an excitation beam from an external lightsource must be brought into the sample so as to excite the intrinsicreporters or extrinsic sensors within the sample/specimen.

There have yet to be any strategies described that avoid the use of anexternal light source (laser, lamp, light emitting diode, etc.) toexcite the luminescent species within the sample/specimen. Furthermore,although a number of strategies exist for developing photonically-basedsensors, there is no strategy for producing the emission and imagingsuch from the sample/specimen without the use of a photon source that isexternal to the sample/specimen.

Several methods involve the idea of using a beta emitter and a liquid,plastic, or inorganic solid scintillator to fabricate photonically-basedinstruments wherein the light source requires no electrical power. Forexample, it has been shown how to use a beta emitter, ⁹⁰Sr, and a liquidor plastic scintillator in concert with time-correlated single photoncounting as a pulsed photon source for the determination ofexcited-state fluorescence lifetimes. It is also known how to producesingle element quenchometrically-based macrosensors for a singleanalyte. Microsensor arrays for the simultaneous detection of multipletarget analytes in a sample wherein the light source was based on a noelectrical power ⁹⁰Sr beta emitter are also known.

In one embodiment, the present invention provides a system which, whenintroduced into a sample, produces emission in a pattern correspondingto the spatial distribution of an analyte, without the presence of apower source or external photon source. Provided by the presentinvention are chemically responsive nanosensors with integrated lightsources. This embodiment of the invention current invention is hereafterreferred to as a chemically responsive nanosensor with an integratedlight source (CRNILS) (FIG. 8 top left). It is a nanosensor particlewhich can be introduced into a sample or specimen. A CRNILS isstructured around one or more beta emitter emitter cores. Beta emittercores which can be used in the photonic sensor described above can alsobe used in the CRNILS (e.g., ³H, ¹⁴C, ²²Na, ³²Si, ³²P, ³⁵S, ³⁶Cl, ⁴¹Ca,⁵⁷Co, ⁶⁰Co, ⁶³Ni, ⁸⁹Sr, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Tc, ¹²⁹I, ¹³⁷Cs, ¹⁴⁷Pm, ¹⁵¹Sm,²⁰⁴Tl, ²¹⁰Pb, ²¹⁰Bi, ²³⁷U, ²³⁸Np, or ²⁴¹Pu. These emitters arepreferably sealed within a material such as ceramic, such that thesample is not needlessly exposed to beta emission or contaminated withcore material. Macroscopic emitters are commercially available from AEATechnology, QSA Incorporated (Burington, Mass.).

The layered structure of the nanosensor facilitates an operation whichsimilar to the operation of the photonic sensor embodiment. Thenanosensor comprises a sensor layer which encases the beta emitter core,as well as an optional layer of scintillation material which is betweenthe core and the one or more sensor layers. Either beta particles fromthe core or beta-stimulated radiation impinge upon the luminophoreswithin the sensing layers (FIGS. 8 and 9) and these species emit and/orabsorb in a manner which is dependent on whether or not they are exposedto a desired analyte. The choices of scintillation material and sensorlayer materials are generally dictated by the same constraints asoutlined above for the photonic sensor. The CRNILS invention can be usedwith luminescence-based sensor strategy that can be coated as a thinfilm sensing layer.

The nanosensor embodiment may optionally comprise a “directing layer,”which can cause the nanosensor to preferentially locate near or withincertain regions of a sample or specimen. Examples of directing layersinclude antibodies, DNA, RNA, enzymes, peptides and related species orother natural and/or synthetic species that have selective interactions(ionic, H-bonding, π-π, Van der Waals, etc.) with a particularsite/domain within the sample/specimen.

A cross sectional view of an example of the invention is presented inFIG. 8. A CRNILS (top left) consists of a nanoscopic sealed beta emitter(e.g., ⁹⁰Sr) that is surrounded by a thin layer of an optionalscintillator layer, one or more sensing layers that contain one or morephotonically active sensor molecules, and an optional directing layer.

The sensing layer can be formed on the face of the scintillator, withinthe scintillator, on the face of the non-scintillating material, orwithin the non-scintillating material as shown in FIG. 9. Strategies forproducing such luminescence-based sensing layers are well-known.Examples are the subject matter of U.S. patent applications Ser. Nos.11/006,857 and 11/031,318, incorporated herein by reference.

In operation one or more CRNILS for specific target analytes are mixedor injected into the sample/specimen. They distribute within thesample/specimen based on the directing layer chemistry (specific ornon-specific/random). The resulting emission (or absorbance) from theCRNILS is/are detected by an external array-based photon detector. Thearray detector allows imaging of the multitude of CRNILS that could belocated with a given sample/specimen.

The chemically-responsive emission from the nanosensors can be collectedby a microscope objective and detected by a suitable array detector(charge transfer device, video camera, or a complementary metal oxidesemiconductor). The signals from the photodetector can also be processedby on-chip electronics or within a computer, or other methods known inthe art.

An advantage of this invention over existing technologies is that anexternal source of photons is not necessary. Excitation of the sensinglayer only comes from within the nanosensor. Excitation of differentluminophores that may high grossly different absorbance/excitationspectra within the sensing layers can be carried out in real time.Long-term monitoring can be achieved depending on the source of the betaemissions (e.g., the half lives of ⁹⁰Sr are 28.5 yrs, ³²P is 14.3 days,and ²³⁸Np is 2.1 days). The noise magnitude in these nanosensors shouldbe much less in comparison to any external light source-based methods.The emitters are intrinsically pulsed at a high rate. This allowslifetime-based sensing to be exploited. It is possible to exploitsteady-state and time-resolved luminescence anisotropies if theexcitation from the emitter and/or scintillator is polarized enough.There is no risk of radioactive contamination from the beta emittersince it is bound in a ceramic matrix and encapsulated. The size of thenanosensors used in the above embodiment of the present invention islimited only by application. For example, if they are to be introducedby injection, they should be small enough to pas into the sample via asyringe or other injection device.

The present invention also provides tailored pins for high densitymicroarray production. The word “array,” as used here, means atwo-dimensional arrangement of elements. In the current context, anarray is formed on a planar or curved support. The array provides a wayof detecting, matching, quantifying, and/or screening known and unknownmolecules based on, among other possible schema, the intermolecularinteractions between the array elements (spots) and the molecules in thesample.

The sensor arrays which can be formed by the method of the presentinvention make use of a wide variety of molecular recognition systems(e.g., ion-cryptan, ion-ligand, host-guest inclusion, DNA base pairing,protein-ligand, protein-protein, antibody-hapten or antibody-antigenbinding) or molecularly imprinted materials. Experiments could involvean enzymatic reaction scheme wherein a colored product is produced orwherein a change in the absorbance and/or emission of chromophore ismodulated by the presence of an analyte as described above. Arrays ofthis type can also be used to screen for lead materials or molecules inapplications ranging from biomaterials to sensing to drug discovery orbiomarker identification.

The size of the array elements (the “feature size”) dictates the elementdensity on the array and thus the number of simultaneous assays,assessments, and/or measurements one can carry out.

One of the main goals in microarray design is to develop new tools todecrease the feature size and increase the array element density (i.e.,the number of sensor elements per unit area). It is also of obviousadvantage if these new tools can be used with existing robotic printers.

Although a number of strategies exist for fabricating microarrays basedon pin printing methods, all current pins suffer from one or more of thefollowing problems: [a] they do not yield feature sizes below 35micrometers, [b] they cannot print all solvents, solutions, and/ormixtures; [c] they are difficult to fabricate; and [d] they areexpensive to purchase.

We have developed a new two part pin system that can be easilyretrofitted to existing robotic pin printers. The system consists of ametallic or polymeric pin mount and a tailored pin. With this system onecan produce features that are on the order of 10 micrometers or less indiameter by using a standard robotic pin printer. The system also allowsone to custom tailor the distal pin tip surface chemistry to allow theprinting of a wide variety of solvents, solutions, and mixtures.

The present invention can be used to increase microarray density by100-fold over standard methods and by 16-fold over the best methods inthe literature.

A cross-sectional view of the invention is given in FIG. 12. The systemconsists of a metallic or polymeric pin holder and a tailored pin. Inoperation, the pin is inserted into the holder such that the distal pinend is directed toward the substrate onto which one wishes to form themicroarray. The pin is then dipped into the solution to be printed. Thesolution wets the pin exterior (solid) or wets the pin exterior andfills the pin channel (channel) depending on the exact pin geometry onechooses (see FIGS. 12 and 13).

The pin is fabricated from non-metallic materials. Examples includeoptical fibers (solid), glass, quartz or organic and/or inorganicpolymer precision rod (solids), or glass, quartz or organic and/orinorganic polymer capillaries (channels); materials that can be easilydrawn after heating. The distal pin end is formed by cutting, etching,or pulling methods. The methods give blunt, point and point endsrespectively.

Pins can be formed by a pulling process on a round or square rod andround or square tubing. A puller like a Warner model PMP-107/PMP-102programmable pipette puller can be used for this task. In operation, therod or tube is loaded into the puller where a point on the rod/tubing isbrought to its softening point through the use of a heating device suchas an electrical filament, laser or gas flame. Once the rod/tubingsoftening point temperature is reached, a mechanical parallel pullingforce is applied to each end of the rod/tube and it is pulled to thedesired tip diameter and profile. Depending on a number of presetvariables (e.g., rob/tubing material, temperature, filament type andpulling force), numerous tip profiles and tip diameters can befabricated. By using this technique tip sizes as small as 0.02 μm can beachieved. Pipette pullers can range in complexity from a simplisticspring/counter weight mechanical puller to highly sophisticatedprogrammable microprocessor controlled pullers.

Once a micropipette has been pulled down to the desired tip diameter andtaper, it can either be used as is or further processed. Processing isdesigned to change tip diameter, tip bevel, tip geometry, bending tovarious angles or to add (fuse) additional segments. To alter (increase)the tip diameter or to apply a bevel to the micropipette the tip issubjected to a controlled micro grinding process using a microbeveller.Microbeveller technology ranges from basic systems using wet aluminaslurry on a rotating mirrored lapping disk, to sophisticated integratedoptical-mechanical diamond based lapping wheel devices.

To reshape the pipette or to fuse the tip into a different geometry, amicroforge system is used. A microforge uses a heating element,microscope, illumination, micromanipulators and microtools to change theshape of or contact fuse other elements to the micropipette pipette byheating certain sections of the micropipette to either soften (forbending) or melting (fusing) the micropipette. Some types of microforgescan also fabricate metal and glass microtools.

The pin surface chemistry, the pin's geometry, the pin channeldimension, the pin distal end dimension, the pin velocity toward thesubstrate, and the pin's contact time to the substrate all effect thefeature size and the ability to form features with a given solution on agiven substrate (M. Schena in Micorarray Analysis, Wiley-Liss, 2003).The pin velocity and contact time are controlled by the robotic pinprinter per se. The other factors are pin-dependent features that dependin large part on the pin's surface chemistry.

One of the difficulties in prior array printing systems is the inabilityto form extremely small sensors due to the failure of the tips to havechemical affinity for the sensor material. In the present invention, thepin surface chemistry can be easily controlled by [i] choosing specificmaterials from which to form the pins (glass, fused silica, organicpolymers) that have intrinsically different surface chemistries and/or[ii] modifying the surface of a particular pin (exterior and/orinterior) with one or more well-known surface derivitization ormodification methods (e.g., silanization, plasma treatment, treatmentwith silicon alkoxides). The pins can be fabricated wherein the surfacechemistry is formed as a gradient along the pin's long axis. Inaddition, the surface chemistry within the channel (gradient or not) canbe different along the pin's long axis in comparison to the pin'sexterior. In many cases, a pin's surface chemistry can be repeatedlychanged.

An example of a reversible modification of the pin surface is thefollowing based on a glass pin and aminopropyltrimethoxysilane (APTES,(EtO)₃Si—(CH₂)₃—NH₂). Here, a clean pin (HCl, NaOH wash) is immersed ina 3% solution of APTES in toluene. The pin and solution are allowed toreact under ambient or reflux conditions for up to 48 h. The APTESreacts with the surface Si—OH residues on the glass pin to form siloxanebonds (Si—O—Si). After a toluene rinsing step, one is left with a pinsurface that consists of (Si—O)₃—Si—(CH₂)₃—NH₂. The Si—(CH₂)₃—NH₂residue can be readily removed by a simple acid/base washing step. Thesurface can then be remodified by using another organically modifiedsilane or chlorosilane with one or more non-hydrolyzable (e.g., Si—C)residues.

An example of a permanent modification of the pin surface is thefollowing based on a glass pin and trimethylchlorosilane (TMCS,Cl—Si(Me)₃). Here, a clean pin (HCl, NaOH wash) is immersed in a 3%solution of TMCS in toluene with 1-2% pyridine. The solution is refluxedfor up to 24 h. The TMCS reacts with the surface Si—OH residues on theglass pin to form Si—O—Si bonds. The pyridine is used to capture the HClthat is produced in the reaction as a pyridine/HCl insoluble salt. Aftera toluene rinsing step, the pin surface consists of Si—O—Si(Me)₃residues. These residues can also be hydrolyzed as described above forthe APTES case, but the required conditions are more aggressive.

Once the pins are formed, they are grouped together in a pin printingapparatus. The pin printing apparatus can comprise pins which havesimilar or identical surface chemistries or geometries. However, thepresent invention also contemplates the use a pin printing apparatus ofpins in a printer wherein the surface chemistry of each pin and/or itsgeometry are different. In this way, multiple solutions/samples withvery different physicochemical properties can be printed simultaneously.

The instant embodiment allows smaller features within microarrays to beprinted by using existing robotic pin printers. Pins can be formed withprinting end bores in the range of from 0.02 to 100 microns which arecapable of printing sensors in the range of from 0.02 to 100 micronsThis leads to a 16 fold improvement in feature density in comparison tothe state-of-the-art and a 100-fold improvement over arrays formed withrobotic pin printers and current commercial pins. The present inventionmakes possible the formation of microarrays having sensor densities of1000 sensors per square millimeter and greater.

The present embodiment also provides a convenient way to reversibly tunethe surface chemistries at or within the pin to fine tune the printing.The current embodiment provides a convenient way to print circular andsquare features. The current embodiment allows for simultaneous printingwith pins having different surface chemistries, allowing thesimultaneous printing of different solutions.

The present invention also provides diversified xerogel-based sensorswhich can conveniently be used with the photonic sensor system of thepresent invention to improved detection and quantification of analytes.

Traditional sensing methods have focused on the development of singlesensors for a given target analyte. Sensor array technology has helpedto improve on this situation by opening the door to the paralleldetection of multiple analytes in a sample. However, individual sensorelements within an array are generally designed for a specific analyte.Thus, the single analyte-single sensor approach, despite paralleldetection schema, remains a fixture in modern chemical sensing.

A persistent problem with the single sensor-single analyte approach isthat in order to ensure accurate results and determine false readingsone must include frequent recalibration and sensor testing steps inorder to ensure that accuracy and sensitivity to the analyte has notdeclined.

The diversified analyte detection embodiment of the present inventionhas significant advantages in that it improves overall sensor accuracy,minimizes the need for recalibration and makes possible the clearidentification of false readings from failing sensor elements.

The diversified sensor embodiment of the present invention provides forthe use of multiple different photonic sensors in a single photonicarray, each sensor being designed to give a different response to atarget analyte (e.g, 0₂). By “different response,” it is meant that eachof the sensors has a different sensor response curve. A “sensor responsecurve” is the relation between sensor response (i.e., reduction orincrease in emission from the sensor upon exposure to givenconcentration of analyte) and analyte concentration. The informationfrom multiple sensor response curves is used to detect and quantify agiven target analyte in a sample.

A non-limiting illustration of the diversified sensor embodiment of thepresent invention is the detection of a specific target analyte, O₂,with xerogel based sensors. The specific detection method is based onquenching of a luminescent molecule in the sensor by O₂.

The sensing of O₂ is a non-limiting example. The invention is amenableto other analytes and other sensing methods. Particularly convenient aremethods that can be performed within a xerogel-based sensor platform.For example, other sensing strategies which can be employed in thecontext of the diversified sensors embodiment include, for example,sensors based on organic polymers, biosensors, sensors based oninorganic polymers, sensors based on molecular imprinting andnanoimprinting, sensors based on molecular recognition or biomimeticstrategies, sensors based on intermolecular interactions of any type(e.g., collisional, electron transfer, energy transfer. The use of thediversified sensors embodiment of the present invention in the sensingof O₂ is described in Example 3.

In order to have a sensor system which can indicate in a singlemeasurement that at least one of the sensors in a sensor array system isfailing, the sensor array should include at least two different types ofsensors. When the two types no longer give the same reading when exposedto the same sample (i.e., substantially identical concentrations ofanalyte), it can be determined that at least one type of sensor hasfailed. In order to know in a single measurement which type of sensorhas failed, it is preferable to include no less than three differenttypes of sensors in the sensor array. If it is determined that one typeof sensor gives a response which is inconsistent with the responses ofthe remaining types of sensors when exposed to the same sample (i.e.,substantially identical concentrations of analyte), the sensors of thattype can be replaced, or their readings can be disregarded. By“consistent response,” it is meant that for a given sensor type, theresponse corresponds to an analyte concentration which is consistentwith the concentrations indicated by the other sensor types.

In another embodiment, the diversified sensors method of the presentinvention can be used to detect multiple analytes, as long as the sensorarray bears at least two different types of sensor corresponding to eachanalyte. In yet other embodiments, the sensors are not arranged in anarray, but are in other arrangements, such as multilayer architectures,continuums, gradients or combinations of these. For example, thediversified sensors embodiment can also be used to improve the accuracyof concentration measurement in imaging applications which measure thespatial variation of an analyte concentration. However, if thediversified sensors embodiment is included in an imaging context, careshould be taken to ensure that any differences in concentrationregistered by different types of sensors which are being compared foraccuracy purposes are not entirely due to spatial variations in analyteconcentration.

In yet another embodiment, the present invention provides high stabilityXerogel-based O₂ sensors with linear calibration curves. These novelsensors exhibit excellent selectivity and sensitivity. Additionally, thesensors have detection limits as low as 1 pg/mL they are easy tocalibrate, and they can be stable for periods at least as long as 12months, and often as long as several years.

In the chemical sensing literature, O₂ is a common target analytebecause it is important in biological, environmental, industrialapplications. O₂ is traditionally quantified by using Clarke electrode(CE); however, the CE consumes O₂ and it can be poisoned by sampleconstituents (e.g., H₂S, proteins, certain anesthetics). Given theselimitations, researchers have expended substantial effort to developoptically based O₂ sensors. The most common of these sensors exploit thewell-known effects of O₂ quenching on the intensity (I) or excited-statelifetime (τ) of an immobilized luminophore (fluorophore or phosphore) asdescribed in Eqn. 1.

$\begin{matrix}{\frac{I_{0}}{I} = {\frac{\tau_{0}}{\tau} = {{1 + {K_{SV}\lbrack Q\rbrack}} = {1 + {k_{q}{\tau_{0}\lbrack Q\rbrack}}}}}} & (1)\end{matrix}$

When luminophores are immobilized, as they are in most available typesof chemical sensors, Eqn 1 is generally not obeyed, and the Stern-Volmerplots for these systems are nonlinear. While the degree of nonlinearitydepends on a wide variety of factors, the nonlinearity in every case isassociated with the luminophore molecules being distributed between twoor more different types of environment which each of which cause theluminophore to exhibit different kq or τ₀ values.

Because the Stern-Volmer plots are nonlinear, currently availablesensors require that one calibrate the sensor using multiple standards(i.e., multipoint calibration). The more non-linear the Stern-Volmerplot, the more the greater the number of standards that are needed toaccurately define the calibration/working curve. Linear Stern-Volmerplots do not require such elaborate standardization/calibration. Inaddition, in some cases, the sensor response is not stable over time.Thus both the multipoint calibration requirement and drift problems canfrustrate the development of a reliable sensor platform.

Over the years, many methods have been described to immobilize sensorchemistries for sensor development. These methods include physisorption,covalent attachment, and entrapment/sequestration. Physisorption methodsare the most simple; however, several disadvantages exist. Randomorientation of the sensing chemistry on the substrate can lead to targetanalyte inaccessibility or distributions of accessibilities. Inaddition, because this method lacks covalent chemical bonds, the“immobilized” sensing chemistry often leaches/desorbs, causing driftproblems. Certain lifetime-based sensor strategies can address some ofthese issues, but these approaches require more costly and complexinstrumentation. Covalent attachment strategies eliminate the leachingproblem; however, they are chemically more complex, they tend to be moretime-consuming and costly to use. Furthermore, due to surfacereorganization, denaturation of protein-based sensing chemistries, orboth, they do not guarantee full accessibility or absence of drift.

Sequestration of the recognition chemistry within a porous,three-dimensional network has become an attractive means to immobilizesensing chemistries. Sol-gel processing methods have been used toalleviate several of the aforementioned immobilization problems. Therehave been a significant number of luminescence-based O₂ sensors reportedin the literature based on sol-gel-derived materials. However, a commonfeature of previous sol-gel-derived O₂ sensors is that the Stern-Volmerplots are non-linear, necessitating multipoint calibration; the sensorresponse is not stable over the long term; or both. Additionally, thesurface structure of many of these sol-gel-derived sensors is oftencracked The cracked surfaces often flake off or the sensor response isneither stable nor reliable.

In this embodiment of the present invention, a series ofluminophore-doped xerogels composed of n-octyltriethoxysilane(Octyl-triEOS) and tetraethyoxysilane (TEOS) are provided. Theluminophore is tris (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)([Ru(dpp)₃]²⁺) and the target analyte is O₂. Results show that thesecomposite xerogels can be tailored to produce uniformly composed sensorsthat exhibit high sensitivity, long-term stability, and linear responsecurves, and a resistance to cracking.

The sensor materials of the present embodiment have an Octyl-triEOScontent in the range of from 0 to 50 mole %, a TEOS content in the rangeof from 50 to 100% mole % and a [Ru(dpp)₃]²⁺ content in the range offrom 1-1000 ppm. Other xerogel formulations and compositions can be usedto tailor the sensitivity and selectivity of the sensors.

In an additional embodiment, the present invention provides a method ofirradiating a sensor having a non-linear Stern-Volmer plot (multipleluminophores, multiple luminophore environments, or both), such that thesensor emits radiation which, upon analysis, gives the same informationgiven by a diversified group of sensors, each sensor containing a singleluminophore in a single environment (i.e., each sensor having a linearSV plot).

We describe a method that uses a single luminescence-based sensor, amodulated excitation source, and phase-sensitive detection to produce awide variety of response profiles from a single sensor. That is, we cancreate response profile diversity from a single sensor. The method isderived and described as follows.

In a luminescence-based quenchometric sensor with one luminophoredistributed so that each luminescent molecule encounters an identicalmicroenvironment, one can write the sensor's response as:

I ₀ /I=τ ₀/τ=1=K _(SV) [Q]=1+k τ ₀ [Q]  (1)

In this expression, I₀ is the intensity from the sensor in the absenceof quencher, Q; I is the intensity in the presence of quencher Q; τ₀ isthe excited-state luminophore lifetime in the absence of Q; τ is theexcited-state luminophore lifetime in the presence of Q; K_(SV) is theStern-Volmer constant; [Q] is the concentration of quencher; and k isthe bimolecular quenching constant the describes the encounters betweenQ and the luminophore. Under these conditions, a plot of I₀/I vs. [Q]will be linear with a slope of K_(SV). To tune K_(SV) (i.e., creatediversity) requires that one adjust k and/or τ₀, making a new sensorelement.

Consider a second quenchometric sensor element of similar design to theone described above, but with two different luminophores (A and B) thateach responds to a different degree to Q. Consider also that theseluminophores possess unique excited-state luminescence lifetimes in theabsence of Q (i.e., τ_(0,A) and τ_(0,B)).

Under steady-state detection conditions one can write this sensor'sresponse as:

I ₀ /I=<τ ₀>/<τ=[(L _(A)/(1+K _(SV,A) [Q]))+[(L _(B)/(1+K _(SV,B) [Q]))  (2)

In this expression, I₀, I, and [Q] are defined above; < > denotes theweighted mean excited-state luminescence lifetimes; K_(SV,A) andK_(SV,B) are the Stern-Volmer constants associated with luminophore Aand B, respectively; and L_(A) and L_(B) are the fractions of the totalluminescence arising from luminophore A and B, respectively. Inaddition, one can write:

K_(SV,A)=k_(A) τ_(0,A)   (3a)

K_(SV,B)=k_(B) τ_(0,B)   (3b)

where k_(A) and k_(B) represent the bimolecular quenching constants forluminophore A and B, respectively. Under these conditions, a plot ofI₀/I vs. [Q] will be non-linear and curve toward the Q axis. To tunethis type of sensor's response (i.e., to create diversity) requires thatone adjust k_(A), τ_(0,A), k_(B), τ_(0,B), L_(A) and/or L_(B), making anew sensor element.

The aforementioned cases can be extended to N luminophores. In all casesthat use steady-state detection methods, the generation of sensorresponse diversity requires that one adjust some aspect of the sensor(vide supra) and fabricate additional sensors.

If we reconsider the A and B containing sensor element that we justdescribed and we now excite the sensor with sinusoidally modulated lightat frequency (f), we see that the resulting luminescence will be phaseshifted (θ) and demodulated (M) to an extent that depends on theluminophore excited-state fluorescence lifetimes and their relativecontributions to the total emission. Specifically, for luminophores Aand B we can write:

θ_(A)=arctan(2πf τ _(A))   (4a)

θ_(B)=arctan(2πf τ _(B))   (4b)

M _(A)=[1+(2πf)² τ_(A) ²]^(−1/2)   (5a)

M _(B)=[1+(2πf)² τ_(B) ²]^(−1/2)   (5b)

We can also write, at any quencher concentration:

L _(A)(Q)=F _(A,0)(1+(k _(A)τ_(0,A) [Q])⁻¹   (6a)

L _(B)(Q)=F _(B,0)(1+(k _(B)τ_(0,B) [Q])⁻¹   (6b)

τ_(A)(Q)=(1/τ_(0,A) +k _(A) [Q])⁻¹   (7a)

τ_(B)(Q)=(1/τ_(0,B) +k _(B) [Q])⁻¹   (7b)

If we record the luminescence from a sensor that contains twoluminophores (A and B) with a phase-sensitive detector (e.g., a lock-inamplifier), we can write the generalized phase-sensitive luminescencesignal (PSLS) as:

PSLS(θ_(D))=L _(A) M _(A) cos(θ_(D)−θ_(A))+L _(B) M _(B)cos(θ_(D)−θ_(B))   (8)

In this expression, L_(A) and L_(B) denote the fraction of the totalluminescence signal that arises from luminophores A and B, respectively;M_(A) and M_(B) represent the demodulation factors associated withluminophores A and B, respectively; θ_(D) is the detector (i.e., lock-inamplifier) phase angle; and θ_(A) and θ_(B) are the phase angles forluminophores A and B, respectively.

If we now use Eqns. 4-7 and substitute them accordingly into Eqn. 8, wecan write an expression for the phase-sensitive analog of Eqn. 2,namely:

PSLSo(θ_(D),0)/PSLS(θ_(D) ,Q)=[L _(0,A) M _(0,A) cos(θ_(D)−θ_(0,A))+L_(0,B) M _(0,B) cos(θ_(D)−θ_(0,B))]/[L _(A)(Q) M_(A)(Q)cos(θ_(D)−θ_(A)(Q))+L _(B)(Q) M _(B)(Q)cos(θ_(D)−θ_(B)(Q))]  (9)

Eqn. 9 provides the key relationship between the fundamental propertiesof the sensor element (i.e., k_(A), T_(0,A), k_(B), T_(0,B), L_(0,A)and/or L_(0,B)) and the phase-sensitive analog of I₀/I (i.e.,PSLS₀/PSLS) as a function of [Q], θ_(D) and f.

FIG. 25 presents the simulated I₀/I vs. [Q] plot for a sensor elementthat contains two luminophores with excited-state lifetimes of 5000 nsand 500 ns (T_(0,A), and T_(0,B), respectively) as a function of addedO₂ (0-100%), the quencher, with L_(A)=L_(B), and k_(A)=k_(B)=3×10⁶(O₂%)⁻¹. The response curve is non-linear. Diversification is possiblehere only by changing k_(A), T_(0,A), k_(B), T_(0,B), L_(A) and/orL_(B), making a new sensor element and corresponding response profile.

FIGS. 26-31 present partial results from a series of simulations basedon the same sensor element described in FIG. 1 when the sensor elementis excited with sinusoidally modulated light and the luminescence isrecorded by phase-sensitive detection. The simulations were performed asa function of [Q], θ_(D) and f FIG. 2 presents the simulation when f=5kHz. FIG. 3 presents the simulation when f=10 kHz. FIG. 4 presents thesimulation when f=20 kHz. FIG. 5 presents the simulation when f=50 kHz.FIG. 6 presents the simulation when f=100 kHz. FIG. 7 presents thesimulation when f=250 kHz. Inspection of these results shows a number ofinteresting trends. First, the response profiles depend on θ_(D).Second, the response profiles depend on f. These results are to becontrasted with the response profile under steady-state conditions (FIG.25) which is singular. Thirdly, there are conditions where thephase-sensitive response profiles exhibit negative excursions. This isan additional unique aspect of our phase-sensitive detection modalitythat is not observed in a steady-state measurement. Together thesesimulations argue for a new strategy to produce massive sensor responsediversity with only a single sensor element.

FIGS. 32-34 present the full 3-D simulations for the sensor elementdiscussed above at 20 kHz (FIG. 32), 50 kHz (FIGS. 33), and 150 kHz(FIG. 34). Inspection of these results echo the trends described in thepartial simulations (i.e., FIGS. 26-31) and they show that there arespecific values of θ_(D) and f that produce hyper-sensitive responsesthat significantly exceed the sensitivity of the same sensor used in asteady-state detection mode. Note too that there are cases where verylarge negative values of PSLS₀/PSLS are indicated; steady-statemeasurements yield only positive values of I₀/I. Finally, notice thatthe general shape of the response curves change significantly dependingon θ_(D) and f. Together, theses results argue for a completely new wayto create diversity in chemical sensing without the need to diversifythe sensors themselves.

FIG. 35 presents a portion of an experiment using a sensor elementcomposed of a sol-gel-derived xerogel that we doped with twoluminophores ([Ru(dpp)₃]²⁺ and [Ru(bpy)₃]²⁺). In this experiment thesensor element is excited with 450 nm light that is modulated at 20 kHz.The emission is passed through an optical filter and detected by aphotodiode. The photodiode output is directed to a lock-in amplifier andthe PSLS is detected as a function of detector phase angle. These curvesare unique and they exhibit most of the features seen in our simulations(FIGS. 26-34).

EXAMPLE 1

The unique nature of the beta emission yields several possible dataformats from the CRNILS (FIG. 10). In FIG. 10A, results are presentedwhere the luminescence spectrum from the sensing layer shifts in thepresence of the target analyte. Potential calibration curves arepresented in FIG. 11A. Curves 1-3 in FIG. 11A represent differentsensing chemistries/sensing layer formulations for the same targetanalyte—diversification. In FIG. 10B, results are presented where theluminescence intensity from the sensing layer changes in the presence ofthe target analyte. Potential calibration curves are presented in FIG.11B. Curves 1-3 in FIG. 11B represent different sensingchemistries/sensing layer formulations for the same targetanalyte—diversification. In FIG. 10C, results are presented where thetime-resolved intensity decay profiles of the sensing layer change inthe presence of the target analyte. Potential calibration curves arepresented in FIG. 11C. Curves 1-3 in FIG. 11C represent differentsensing chemistries/sensing layer formulations for the same targetanalyte diversification. The sensing modalities presented in FIGS. 10and 11 can be used independently or together. One particularlyattractive aspect of these detection modalities arises becauseexcited-state lifetimes are independent of the luminophoreconcentration. Therefore, if there were some level of photobleaching,decrease in source output, etc., it would not present a problem.Further, such lifetime-based sensing strategies simplify calibration.)

EXAMPLE 2

FIG. 14 presents examples of pin printed microarrays that have beenformed in the laboratory. The printing solution was a 50:50 mole %tetramethylorthosilane:n-octyl-trimethoxysilane-based xerogel doped withthe fluorophore rhodamine 6G. The arrays are illuminated with a laser inan epi-fluorescence geometry, detection is with a CCD camera. The“Pulled fiber” array was formed from a single fused silica optical fiber(geometry, round, solid, points in FIG. 12). The printed feature sizesare 14±3 micrometers in diameter. Arrays are also shown for commercialquill and solid pins. In all cases, the feature size with the commercialpins is at least an order-of-magnitude larger in diameter when comparedto our “Pulled fiber”. The pulled fiber can be used to increase theprinted element density by ˜100×).

EXAMPLE 3

Inspection of Eqn. 1 reveals two strategies for tuning the sensitivityof any quenchometric sensor—adjust τ₀ and/or adjust kq. (Note; In othersensor schemes one might tune the binding affinity, partitioncoefficient, etc. to generate the necessary sensor diversity.)

To illustrate the potential of this tuning we formed a diversifiedlibrary of xerogel-based sensor elements for O₂. Here, each sensorelement is designed so that it exhibits a distinct calibration curveand/or response time course to dissolved or gaseous O₂. In thisparticular example, these diversified xerogel-based photonic sensorlibraries are derived from luminophore-doped xerogels. The xerogels arccomposed of n-alkyl (C₁-C₁₂)-triethoxysilane (C₁-C₁₂)-TriE0S) andtetraethyoxysilane (TEOS) or n-alkyl (C₁-C₁₂)-trimethoxysilane(C₁-C₁₂)-TriMOS) and tetramethyoxysilane (TMOS) precursors. Theluminophores are tris(4.7-diphenyl-1,10-phenanthroline)ruthenium(II),[Ru(dpp)₃]²⁺ and/or tris(2,2′-bipyridyl)ruthenium(II), (Ru(bpy)₃]²⁺ Byusing these precursors and luminophores we adjust τ₀ and kq at will toproduce collections of sensors for the same target analyte with uniqueresponses (i.e., diversification).

Other xerogel precursors and luminophores can be used to further tunethe sensor's analytical figures of merit beyond the ranges presentedhere. Secondary dopants (e.g., oligomers, polymer, surfactants, lipids,organically modified silanes) can also be added to the xerogel to effectfurther diversity. Other detection and molecular recognition schemes(e.g., molecular imprinting, nanoimprinting, SSTTX, PIXIES, affinity,protein-ligand, protein-protein, hybridization) can also be used.

FIG. 15 presents a series of false color images from a small segment ofa diversified xerogel-based O₂ responsive sensor library as a functionof the gaseous O₂ concentration. This library is illuminated with anexternal laser and the emission from the individual sensor elements isdetected by a charge coupled device detector. [Note: Otherexcitation/detection approaches are also possible]. The individualpanels show the O₂-dependent response from a 25 element, diversifiedsensor library. Each column (labeled Sensors A-E) represents one of fivesensor types. The five elements within each column represent replicatesof a given sensor type. The individual sensor types were fabricated froma binary xerogel that is composed of identical molar ratios of TEOS andC₈-TriEOS precursors doped with different molar ratios of [Ru(dpp)₃]²⁺and [Ru(bpy)₃]²⁺, The excited-state lifetimes of [Ru(dpp)₃]²⁺ and[Ru(bpy)₃]²⁺ differ by 10-fold, allowing us to tune the sensor response(τ₀ tunability per Eqn. 1). [Note: Since we are forming xerogels thatare doped with two luminophores that exhibit different τ₀ values, it isnot rigorously correct to use the term τ₀. To be rigorous one should usethe term <τ₀> (=f_(dpp) τ_(0dpp)+f_(dpy) τ_(0dpy)). In this expression,f_(x) represents the fraction of the total intensity arising fromluminophore x (dpp or bpy) and τ_(0x) is the excited-state luminescentlifetime of luminophore x (dpp or bpy) in the absence of quencher.]

The actual response curves from these five sensors, as a function ofgaseous and dissolved O₂, are presented in FIGS. 16A and 16B,respectively. The differential response is obvious as is the fact thatthe xerogels that contain both [Ru(dpp)²⁺]+ and [Ru(bpy)₃j²⁺ (SensorsB-D) exhibit non-linear Stern-Volmer plots.

FIG. 17 presents the response profiles from a portion of anotherdiversified O₂ responsive sensor library. Here, we show results fromeight sensors each designed for O₂ as a function of added gaseous O₂.These specific sensors were fabricated from a binary xerogel composed ofidentical molar ratios of TEOS and C₁-C₁₂-TriEOS or TMOS andC₁-C₁₂-TriMOS. The luminophore is [Ru(dpp)₃]²⁺. The [Ru(dpp)₃]²⁺excited-state luminescence lifetime is independent of the xerogelcomposition; however, the bimolecular quenching constant within thesexerogel composites differ by 10-fold. Thus, we can tune the sensorresponse (kg tunability per Eqn. 1).

FIGS. 15-17 illustrate the diversified xerogel-based analyte sensingstrategy. Tables 1 and 2 show the improvement in accuracy realized byusing this diversified sensor scheme.

TABLE 1 Comparison of individual and collective sensor accuracy forgaseous O₂. Sensor Unknown = 45% O₂ Unknown = 85% O₂ A 48.0 ± 3.3 80.4 ±3.3 B 46.6 ± 5.1 86.9 ± 7.7 C 44.0 ± 22  82.6 ± 7.6 D 44.4 ± 1.7 84.6 ±4.5 E 45.4 ± 1.9 82.3 ± 3.0 All 5 Sensors 45.4 ± 1.9 ~3.3 ± 2.5

TABLE 2 Comparison of individual and collective sensor accuracy fordissolved O₂. Sensor Unknown = 40% O₂ Unknown = 80% O₂ A  44.1 ± 10.3 79.1 ± 11.9 B 43.6 ± 2.8 83.2 ± 2.5 C 40.2 ± 2.2 81.8 ± 7.2 D 41.5 ±3.2 80.1 ± 5.4 E 39.1 ± 1.6 78.1 ± 2.9 All 5 Sensors 41.7 ± 2.2 80.4 ±2.0

EXAMPLE 4

Chemical Reagents.Tris(4,7′-diphenyl-1,10′-phenanathroline)-ruthenium(II) chloridepentahydrate was purchased from GFS Chemicals, Inc. and purified asdescribed in the literature. TEOS and Octyl-triEOS were purchased fromUnited Chemical Technologies. HCI was obtained from Fisher ScientificCo. EtOH was a product of Quantum Chemical Corp. All reagents were usedas received unless mentioned otherwise. Deionized water was prepared toa specific resistivity of at least 18 MΩcm by using a Barnstead NANOpureII system.

Preparation of [Ru(dpp)₃]²⁺-Doped Octyl-triEOS/TEOS Composite XerogelSensing Films. A pure TEOS-derived sol was prepared by mixing TEOS(3.345 mL, 15 mmol), water (0.54 mL, 30 mmol). EtOH (3.4 mL, 60 mmol),and I-1Cl (15 aL of 0.1 M HCl, 0.0015 mmol). This sol solution was thencapped and magnetically stirred under ambient conditions for 6 h. Thisparticular formulation was chosen because it is representative ofTEOS-based xerogels reported in the literature

The Octyl-triEOS/TEOS composite sols were prepared by mixing TEOS andOctyl-triEOS (6.5 mmol in total) together to form solutions thatcontained 20, 40, 50, 60, or 80 mol % Octyl-triEOS. These particularprecursors were selected to provide a wide range of physicochemicalproperties in the final xerogels. To each of these sol solutions weadded EtOH (1.25 mL, 22 mmol) and HCI (0.4 mL of 0.1 N HCl, 0.04 mmol).These solutions were then capped and magnetically stirred under ambientconditions for I h. These solutions were then diluted 1:1 (v/v) withEtOH. We found that the EtOH dilution step was necessary to lower theoverall solution viscosity, slow the onset of gelation, and improve thefinal spin-coated film quality.

The luminophore-doped sol solutions were prepared by mixing 60 μL of 2mM [Ru(dpp)₃]²⁺ (in EtOH) with 540 4 of the corresponding sol solution(vide supra). Blanks were prepared by omitting the [Ru(dpp)₃]²⁺ Thesesol mixtures were capped and magnetically stirred under ambientconditions for 10 mm prior to spin casting.

Xerogel films were formed by spin casting onto 2.5 cm×2.5 cm glassmicroscope slides. Each slide was first cleaned by soaking in 1 M NaOHfor 24 h. All slides were rinsed with copious amounts of deionized waterand EtOH and dried under ambient conditions. Films were formed bydelivering 100 4 of a given sol solution onto a glass slide placed inthe spin coater. The spin coater was then engaged and the rotationalvelocity adjusted to 3000 rpm. Spinning was continued for 30 s. Allfilms were stored in the dark under ambient conditions for the long-termaging studies. The aging time clock begins immediately after a film iscast. Experiments were conducted over an 11-month period.

Profilometry measurements were performed at regular time intervals onfilms that had aged for between 1 week and 11 months. There was no morethan a 10% change in the individual film thickness over 11 months.

Samples and blanks were prepared in triplicate on five separateoccasions by using fresh reagent batches. The average and standarddeviation for all measurements (n=15) are reported. The blankcontribution was <1% of the observed luminescence when the xerogels weresubjected to 100% O₂.

Instrumentation. A Hitachi model S-4000 field emission scanning electronmicroscope (SEM) was used to record the film images. The acceleratingvoltage was maintained at 20 kV.

All steady-state fluorescence measurements were carried out by using anSLM-Aminco model 48000 MHF spectrofluorometer. A xenon arc lamp was usedas the excitation source (λ_(X)=475 nm). The excitation radiationimpinged on the film-coated side of the glass substrates at an incidentangle of ˜60° with a 90° angle maintained between the excitation beamtrajectory and the emission collection optics. The emission wasmonitored through a 570-nm long-pass filter as we gradually adjusted theenvironment surrounding the sample from pure N₂ to pure O₂. We typicallyallow 30 s between changes in the N₂/O₂ concentration to ensure that anew equilibrium point had been established. Equilibrium was evident whenthe luminescence intensity remained constant to within +2%. There was nohysteresis observed. The O₂ concentrations were accurate to ±1%.Response times were <5 s.

The time-resolved intensity decay measurements were performed by usingan N₂-pumped dye laser as the excitation source (Photon TechnologyInternational, model GL-301 dye and model GL-3300 pump). The dye laseroutput was adjusted to 448 nm. The sample emission was passed through a570-nm long-pass filter and detected with a photomultiplier tube(Hamamatsu, model R928). The photomultiplier tube output (terminatedinto 50 Q) was connected to a 200-MI-1z digital oscilloscope (Tektroniz.model TDS 350) that was interfaced to a personal computer. During thesesmeasurements, a pure gas or gas mixture was used to purge the entiresample chamber for 5 mm and 10-20 data sets were collected when thetotal area under a intensity decay profile remained constant (+2%). ACVI LabWindows software program was used to acquire the data. Theintensity decay profiles were analyzed by using SigmaPlot version 3.0(Jandel Scientific). The short instrument response function (−˜−20 ns).combined with the long [Ru(dpp)₃]²⁺ excited-state luminescence lifetime(>3 μs), removes the need for deconvolution.

SEM Images. The surface structure of a sensor platform is generallyimportant to its sensitivity and reliability. FIG. 18 is a series of SEMmicrographs of pure TEOS and Octyl-triEOS/TEOS composite xerogel films.Significant cracking was always observed for the pure TEOS-based xerogelfilms. In contrast, all the Octyl-triEOS/TEOS composite xerogel filmsappear, at this magnification, to be smooth and crack free. Profilometryexperiments showed that the Octyl-triEOS/TEOS composite xerogel filmswere 1.0+0.1 μm thick.

Higher resolution SEM micrographs (FIG. 19) showed that certaincomposite xerogel films (e.g., ≧60 mol % Octyl-triEOS) were notparticularly uniform. The surface of these composite xerogels werecharacterized by two regions: (1) homogeneous and transparent and (2)heterogeneous and opaque. The transparent regions were thicker onaverage and they were characterized by a few small (50-60-nm diameter),uniform raised features on a more or less uniform base. The opaqueregion was much less thick, and it was characterized by a less uniformset of larger (50-200-nm diameter) features on the glass substrate base.

Average Sensitivity and Stability. In FIG. 20, we report the effects ofaging time and xerogel composition on the O₂ sensor's averagesensitivity and response stability. At early times following sensorfabrication, the pure TEOS-based xerogels exhibit the greatestI_(N2)/I_(O2) of any sensor tested. However, as this xerogel ages, thesensitivity decreases by almost 5-fold (I_(N2)/Io₂=19 at 2 weeks andI_(N2)/Io₂=4 at 11 months). Thus, the pure TEOS-based sensor drifts withtime, losing significant sensitivity with time. This type of behaviorhas also been observed with pyrene-doped TEOS-based xerogels.

The Octyl-triEOS/TEOS composite xerogels exhibit sensitivities thatdepend on the mole percent Octyl-triEOS in the xerogel. The greatestsensitivity is seen for those xerogels that contain the higherOctyl-triEOS mole percent. We attribute the increasing sensitivity tothe nonbridging Si—C₈H₁₇bonds that act as network modifiers, increasingthe overall xerogel hydrophobicity and terminating the silicate network.

The sensitivity of the Octyl-triEOS/TEOS composite xerogels areremarkably stable over the course of 11 months. For example, over thecourse of an 11-month stability study, the 20, 40, and 60 mol %Octyl-triEOS composites, exhibit I_(N2)/Io₂ values of 8.99+0.49 (RSD5.5%), 12.06+0.19 (RSD=1.5%), and 16.48+1.14 (6.9% RSD), respectively.For the 50 mol % Octyl-triEOS composites, over the same time period, theI_(N2)/Io₂ was 14.37+0.58 (RSD=4.0%). Together these results show thatsensors based on the Octyl-triEOS/TEOS composite xerogels exhibitexcellent long-term stability and sustained high sensitivity incomparison to a pure TEOS-based xerogel sensor. The Octyl-triEOS/TEOSxerogel films are reportedly more flexible in comparison to the pureTEOS-base films. Given this, we speculate that the observed improvementsin sensor long-term stability and sustained high sensitivity arise fromthe increased flexibility that overcomes the xerogel shrinkage and porecollapse with time.

Stern-Volmer Plots. FIG. 21 presents typical intensity-basedStern-Volmer plots for a randomly selected set of 3-month-old[Ru(dpp)₃]²⁺-doped xerogel films. The solid lines represent the bestfits to the data. The recovered fitting parameters are compiled in Table3.

TABLE 3 Effect of Compositions and Aging Time on the O₂ Quenching of[Ru(dpp)₃]²⁺ - doped Octyl-triEOS/TEOS Xerogels^(a) Octyl-triEOS agingStern-Volmer Lehrer Demas^(c) (mol %) time^(b) K_(SV)(|O₂|⁻¹) r² f_(η)^(d) K_(SVq) ^(#)(|O₂|⁻¹) r² f₁ K_(SV1)(|O₂|⁻¹) K_(SV2) (|O₂|⁻¹) r² 0 3m 0.094 ± 0.008 0.9030 0.92 ± 0.01 0.284 ± 0.040 0.9724 0.76 ± 0.02 1.02± 0.21 0.013 ± 0.001 0.9982 5 m 0.050 ± 0.003 0.9430 0.88 ± 0.01 0.116 ±0.015 0.9702 0.55 ± 0.02 0.68 ± 0.18 0.016 ± 0.001 0.9988 20 6 w 0.086 ±0.003 0.9872 0.97 ± 0.04 0.107 ± 0.025 0.9866 0.36 ± 0.20 5.0 ± 48 0.050 ± 0.014 0.9921 11 m  0.077 ± 0.004 0.9876 0.96 ± 0.03 0.115 ±0.017 0.9873 0.52 ± 0.23 0.69 ± 0.66 0.032 ± 0.007 0.9895 40 6 w 0.110 ±0.003 0.9896 11 m  0.112 ± 0.004 0.9877 50 3 m 0.138 ± 0.002 0.9932 11m  0.126 ± 0.003 0.9904 60 6 w 0.142 ± 0.004 0.9905 11 m  0.156 ± 0.0040.9826 ^(a)When an entry is not given, the Stern-Volmer model is thebest model. ^(b)m, months; w, weeks. ^(c)Terms are from eq 2. ^(d)f_(η),fraction of luminophore that is quenchable. ^(#)K_(SVq), Stern-Volmerquenching constant of luminophore that is quenchable.

Inspection of these results reveal several key points. First, theStern-Volmer plot for the pure TEOS-based xerogel exhibits downwardcurvature and the fit to the Stern-Volmer model (eq 1) is poor(r²=0.9030). Superior fits are achieved for the Leherer and Demasmodels, the Demas model offering the best fit of the three models tested(r^(2=0.9982)). These results are fully in line with the behavior of awide variety of luminophore. doped sol-gel derived xerogels. Second, theStern-Volmer plots for the Octyl triEOS/TEOS composite xerogel films arereasonably well described by eq 1. That is, is it not necessary to usethe more complex Lehrer or Demas models to describe the Stern-Volmerplots for the Octyl.triEOS/TEOS composite xerogels. (Note: Theseparticular 20 mol % Octyl-triEOS data appear to deviate from theStern-Volmer model at low 0₂ concentrations; however. r² does notimprove significantly for the Lehrer or Demas models and the imprecisionin the recovered parameters is always large for these more complexmodels for this xerogel.) Together these results demonstrate that themicroenvironment that surrounds the [Ru(dpp)₃]²⁺ molecules changes frombeing heterogeneous within the pure TEOS-based xerogel to being morehomogeneous within composite xerogels that contain >20% Octyl-triEOS.The linearity of the Stern-Volmer plots open a door to simple two pointcalibration strategies. Third, the recovered K_(8˜) values (Table 3) forthe pure TEOS-based xerogel films decrease as the film age increases.The recovered Ksv values for the Octyl-triEOS/TEOS composite xerogelfilms do not suffer this problem; they exhibit excellent long termstability. This result is entirely consistent with FIG. 20. Finally,inspection of FIG. 21 shows that the error bars associated with the pureTEOS-based xerogel films are 3-10-fold larger in comparison to theOctyl-triEOS/TEOS composite xerogel films. This result demonstrates thatthe film-to-film reproducibility is significantly better for thecomposite xerogels in comparison to the pure TEOS-based xerogels.

In FIG. 22, we report the effects of xerogel composition on the averageStern-Volmer quenching constant, <Ksv>. These results show that <K_(5v)>increases as we increase the mole percent Octyl-triEOS in the xerogel.To explore the origin of the improved sensitivity, we carried out aseries of time resolved intensity decay experiments.

Time-Resolved Intensity Decays. FIG. 23 presents a representative seriesof time-resolved intensity decay traces for 3-month-old [Ru(dpp)₃]²⁺±doped xerogels when they are maintained in a pure N₂ atmosphere alongwith fits to single—and double—exponential decay models. The recovereddecay terms are collected in Table 4.

TABLE 4 Effects of Octyl-triEOS/TEOS Xerogel Composition on theRecovered [Ru(dpp)3]²⁺ Time-Resolved Intensity Decay Kinetics.Octyl-triEOS (mol %) f₁ ^(b) τ₁ (ns) τ₂ (ns) r²  0 1.00 4773 ± 18 0.65850.46 ± 0.01 2741 ± 48 6872 ± 77 0.9967 20 1.00 5660 ± 11 0.9872 0.25 ±0.01 2983 ± 59 6668 ± 32 0.9929 40 1.00 5569 ± 2  0.9993 50 1.00 5645 ±4  0.9982 60 1.00 5453 ± 8  0.9978 ^(a)Samples were aged for 3 months.^(b)f₁ + f₂ = 1.

These results clearly demonstrate that the [Ru(dpp)₃]²⁺ time-resolvedintensity decay is multiexponential in the pure TEOS-based xerogel. Thisis common for luminophores doped in a wide variety of host matrices. Asthe mole percent of Octyl triEOS in the composite xerogel increases, thetime-resolved intensity decay traces become single exponential. To thebest of our knowledge, these represent the first luminophore-dopedxerogels with >20 mol % Octyl-triEOS that exhibit purelysingle-exponential intensity decay traces and linear Stern-Volmer plots(FIG. 21). Taken together, these results argue that the microenvironmentsurrounding the [Ru(dpp)₃]²⁺ molecules is homogeneous within thoseOctyl-triEOS/TEOS xerogel composites that contain more than 20 mol %Octyl-triEOS. The fact that the Stern-Volmer plots appear to be “linear”for the 20 mol % Octyl-triEOS xerogels (Table 3) while the intensitydecay traces are clearly multiexponential is a manifestation of theincreased information content of the time-resolved measurements incomparison to a simple intensity measurement.

The sensitivity of any quenchometric O₂ sensor depends on theStern-Volmer quenching constant, Ksv, which in turn depends on twofactors (eq 1): v₀ and A˜.

FIG. 24 summarizes the effects of Octyl-triEOS mole percent on theaverage τ₀ and k_(q) (i.e., <τ₀> and <k₀>) values for 3-month-oldxerogels. These results show that <to> increases from −′5 μs in the pureTEDS-based xerogel film to ″−6 us for 60 mol % Octyl-triEOS/TEOScomposite, a 20% increase. Thus, the increase in the [Ru(dpp)₃]²⁺luminescence lifetime alone is not entirely responsible for the 50%increase in O₂ sensitivity for the Octyl-triEOS/TEOS composites. Theremaining cause of the increased sensitivity (30%) arises from anincrease in <kq>. Thus, 40% of the total improvement in O₂ sensitivityarises from an increase in [Ru(dpp)₃]²⁺'s excited-state lifetime withinthe Octyl-triEOS/TEOS xerogel composites and 60% comes about from aconcomitant increase in <k,₁> (i.e., O₂ transport within theOctyl-triEOS/TEOS xerogel composites).

Example 4 illuminates the quenchometric behavior of [Ru(dpp)₃]²⁺sequestered within a series of Octyl-triEOS/TEOS composite xerogelfilms. Upon adding Octyl-triEOS to TEDS, a number of changes occur.First, the quality of the films improve from being cracked (pure TEDS)to being uniform and crack free (composites that contain 20-50 mol %Octyl-triEOS). Second, the O₂ sensor long-term stability is improvedsignificantly by adding the Octyl-triEOS component. Over a period of 11months, the sensitivity for a pure TEDS-based sensor drops by 400%whereas a 50 mol % Octyl-triEOS/TEOS composite xerogel remains unchanged(RSD=4%). Third, the average O₂ sensitivity (I_(N2)/Io₂) increases as weincrease the mole percent Octyl-triTEOS within the composite. Incomparison to an 11-month-old TEDS xerogel, a 50 mol % OctyltriEOS/TEOScomposite xerogel exhibits a greater than 4-fold improvement insensitivity. For 3-month-old films, 40% of the observed increase in theobserved O₂ sensitivity comes about from an increase in the[Ru(dpp)₃]²⁺s excited-state lifetime and 60% arises from a concomitantincrease in O₂ transport within the Octyl-triEOS/TEOS xerogel composites(i.e., <kq>). Fourth, based on the Stem-Volmer plots and thetime-resolved intensity decay traces, the [Ru(dpp)₃]²⁺ microenvironmentsurrounding the [Ru(dpp)₃]²⁺ molecules is homogeneous within thoseOctyl-triEOS/TEOS xerogel composites that contain more than 20 mol %Octyl-triEOS.

While the invention has been described with preferred embodiments, it isto be understood that variations and modifications may be resorted to aswill be apparent to those skilled in the art. Such variations andmodifications are to be considered within the purview and the scope ofthe claims appended hereto.

1. A photonic sensor system for sensing an analyte in a sample, saidsystem comprising: a) at least one encapsulated beta emission source; b)optionally, a scintillation layer which radiates upon exposure to betaemission from said beta emission source; c) at least two sensors whichcomprise luminophores capable of radiating upon exposure to betaemission from said beta emission source or radiation from saidscintillation layer, wherein the intensity, polarization, spectrum, orlifetime of said luminophore radiation is changed upon exposure of theat least two sensors to said analyte; wherein the scintillating layer isdisposed between the beta emission source and said at least two sensors.2. A photonic sensor system as in claim 1, wherein said a least twosensors comprise an array of photonically-active sensor elements.
 3. Aphotonic sensor system as in claim 1 wherein the beta emission source isencapsulated within a ceramic or steel matrix.
 4. A photonic sensorsystem as in claim 1 wherein the beta emitter is ⁹⁰Sr.
 5. A photonicsensor system as in claim 1 wherein the photonic sensor system does notinclude a scintillation layer.
 6. A photonic sensor system as in claim1, said at least two sensors having different calibration curves withrespect to said analyte.
 7. A photonic sensor system as in claim 6wherein said at least two sensors are xerogel sensors.
 8. A photonicsensor system as in claim 7 wherein the xerogels comprising said sensorsare different from each other.
 9. A photonic sensor system as in claim 6wherein said at least two sensors comprise luminophores.
 10. A photonicsensor system as in claim 9 wherein said luminophores are different fromeach other.
 11. A photonic sensor system as in claim 7 for the detectionand quantization of oxygen, wherein said at least two sensors comprisetris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II),n-octyltriethoxysilane, and tetraethoxysilane.
 12. A nanosensor particlefor the detection of an analyte, said particle comprised of: a) a betaemitter core; b) optionally, a scintillation layer which radiates uponexposure to beta emission from said beta emission source. c) a sensinglayer comprising luminophores which are capable of radiating uponexposure to beta emission from said beta emission source or radiationfrom said scintillation layer.
 13. A nanosensor particle as in claim 12wherein the outermost layer is a directing layer.
 14. A nanosensorparticle as in claim 12 wherein the nanosensor particle does not includea scintillation layer.
 15. A nanosensor particle as in claim 12 whereinthe nanosensor particle does not include a directing layer, and whereinthe intensity, spectrum, polarization, or lifetime of said luminophoreradiation is changed in the presence of said analyte.
 16. A method forforming a microarray having a sensor density of at least 1000 sensorsper square millimeter, said method comprising: a) forming a pin printingapparatus comprising nonmetallic tubes having printing end bores in therange of from 0.02 to 100 microns; b) printing a microarray comprised ofsensor elements having diameters in the range of from approximately 0.2to 100 microns onto a substrate with said pin printing apparatus.
 17. Amethod as in claim 16, wherein said pin printing apparatus comprisestubes which are pulled tubes.
 18. A method as in claim 16, wherein theprinting ends of said tubes are treated to facilitate the formation ofsaid sensors.
 19. A method as in claim 17 wherein said tubes are glass.20. A method as in claim 18 wherein said tubes are glass.
 21. A photonicsensor system for sensing an analyte in a sample, said systemcomprising: a) a source of sinusoidally modulated radiation; b) at leastone sensor having a non-linear Stern-Volmer plot which comprisesluminophores capable of radiating upon exposure to said sinusoidallymodified radiation; wherein the intensity, polarization, spectrum, orlifetime of said luminophore radiation is changed upon exposure of theat least one sensor to said analyte;